Piperazine CCR-3 receptor antagonists

ABSTRACT

The invention provides compounds of Formula (I): 
                 
 
wherein: R 1 -R 4 , A, L, and X have any of the values defined in the specification that are CCR-3 receptor antagonists, pharmaceutical compositions containing them, methods for their use, and methods and intermediates useful for preparing them.

This application claims the priority benefit under Title 35 U.S.C.119(e) of U.S. Provisional Application No. 60/334,655, filed Nov. 30,2001.

This application also incorporates by reference U.S. Provisionalapplication Ser. No. 60/334,819, “CCR-3 Receptor Antagonists (I),” andU.S. Provisional application Ser. No. 60/334,653, “Piperidine CCR-3Receptor Antagonists,”, both filed concurrently on Nov. 30, 2001, andissued U.S. Pat. No. 6,323,223 and U.S. Pat. No. 6,166,015.

FIELD OF THE INVENTION

The invention relates to certain piperazine derivatives that are CCR-3receptor antagonists, pharmaceutical compositions containing them,methods for their use, and methods and intermediates useful forpreparing them.

BACKGROUND INFORMATION

Tissue eosinophilia is a feature of a number of pathological conditionssuch as asthma, rhinitis, eczema and parasitic infections (see Bousquet,J. et al., N. Eng. J. Med. 323: 1033-1039 (1990) and Kay, A. B. andCorrigan, C. J., Br. Med. Bull. 48:51-64 (1992)). In asthma, eosinophilaccumulation and activation are associated with damage to bronchialepithelium and hyperresponsiveness to constrictor mediators. Chemokinessuch as RANTES, eotaxin and MCP-3 are known to activate eosinophils (seeBaggiolini, M. and Dahinden, C. A., Immunol. Today. 15:127-133 (1994),Rot, A. M. et al., J. Exp. Med. 176, 1489-1495 (1992) and Ponath, P. D.et al., J. Clin. Invest., Vol. 97, #3, 604-612 (1996)). However, unlikeRANTES and MCP-3 which also induce the migration of other leukocyte celltypes, eotaxin is selectively chemotactic for eosinophils (seeGriffith-Johnson, D. A. et al., Biochem. Biophy. Res. Commun. 197:1167(1993) and Jose, P. J. et al., Biochem. Biophy. Res. Commun. 207, 788(1994)). Specific eosinophil accumulation was observed at the site ofadministration of eotaxin whether by intradermal or intraperitonealinjection or aerosol inhalation (see Griffith-Johnson, D. A. et al.,Biochem. Biophy. Res. Commun. 197:1167 (1993); Jose, P. J. et al., J.Exp. Med. 179, 881-887 (1994); Rothenberg, M. E. et al., J. Exp. Med.181, 1211 (1995) and Ponath, P. D., J. Clin. Invest., Vol. 97, #3,604-612 (1996)).

Glucocorticoids such as dexamethasone, methprednisolone andhydrocortisone have been used for treating many eosinophil-relateddisorders, including bronchial asthma (R. P. Schleimer et al., Am. Rev.Respir. Dis., 141, 559 (1990). The glucocorticoids are believed toinhibit IL-5 and IL-3 mediated eosinophil survival in these diseases.However, prolonged use of glucocorticoids can lead to side effects suchas glaucoma, osteoporosis and growth retardation in the patients (seeHanania, N. A. et al., J. Allergy and Clin. Immunol., Vol. 96, 571-579(1995) and Saha, M. T. et al., Acta Paediatrica, Vol. 86, #2, 138-142(1997)). It is therefore desirable to have an alternative means oftreating eosinophil related diseases without incurring these undesirableside effects.

Recently, the CCR-3 receptor was identified as a major chemokinereceptor that eosinophils use for their response to eotaxin, RANTES andMCP-3. When transfected into a murine pre-beta. lymphoma line, CCR-3bound eotaxin, RANTES and MCP-3 conferred chemotactic responses on thesecells to eotaxin, RANTES and MCP-3 (see Ponath, P. D. et al., J. Exp.Med. 183, 2437-2448 (1996). The CCR-3 receptor is expressed on thesurface of eosinophils, T-cells (subtype Th-2), basophils and mast cellsand is highly selective for eotaxin. Studies have shown thatpretreatment of eosinophils with an anti-CCR-3 mAb completely inhibitseosinophil chemotaxis to eotaxin, RANTES and MCP-3 (see Heath, H. etal., J. Clin. Invest., Vol. 99, #2, 178-184 (1997). Applicants' issuedU.S. patents U.S. Pat. Nos. 6,140,344 and 6,166,015 and published EPapplication EP903349, published Mar. 24, 1999 disclose CCR-3 antagoniststhat inhibit eosinophilic recruitment by chemokine such as eotaxin.

Therefore, blocking the ability of the CCR-3 receptor to bind RANTES,MCP-3 and eotaxin and thereby preventing the recruitment of eosinophilsshould provide for the treatment of eosinophil-mediated inflammatorydiseases.

SUMMARY OF THE INVENTION

The present invention concerns novel piperazine derivatives which arecapable of inhibiting the binding of eotaxin to the CCR-3 receptor andthereby provide a means of combating eosinophil induced diseases, suchas asthma.

In a first aspect, this invention provides a compound of Formula (I):

wherein:

R¹ is (C₁-C₂)alkylene;

R² is optionally substituted phenyl;

R³ is hydrogen, alkyl, acyl, aryl, or arylalkyl;

ring A is a cycloalkyl, heterocyclyl, or optionally substituted phenyl;

L is —C(═O)—, —C(═S)—, —SO₂—, —C(═O)N(R_(a))—, —C(═S)N(R_(a))—,—SO₂N(R_(a))—, —C(═O)O—, —C(═S)O—, —S(═O)₂O—;

-   -   where R_(a) is hydrogen, alkyl, acyl, aryl, arylalkyl,        alkoxycarbonyl, or benzyloxycarbonyl;

X is absent, —(CR′R″)O—, —(CR′R″)S—, —(CR′R″)NR_(b)— or alkylene;

-   -   where R′ and R″ are independently hydrogen or alkyl, and R_(b)        is hydrogen or alkyl; and

R⁴ is aryl or heteroaryl;

provided the compound of Formula I is not1-{2-[4-(3,4-dichlorobenzyl)piperazin-1-yl]cyclohexyl}-3-(3-methoxyphenyl)urea;and

provided that when ring A is phenyl or cyclohexyl, then R² issubstituted phenyl;

and prodrugs, individual isomers, racemic and non-racemic mixtures ofisomers, and pharmaceutically acceptable salts and solvates thereof.

More preferably for the compound of Formula I, R⁴ is not alkoxyphenylwhen R¹ is methylene; R² is 3,4-dichlorophenyl, R³ is hydrogen; ring Ais cyclohexyl; L is C(═O)N(R_(a))—; X is absent; and R_(a) is hydrogen.

Even more preferably for the compound of Formula I, A is not cyclohexylwhen L is C(═O)N(R_(a))—; X is absent; and R_(a) is hydrogen.

In a second aspect, this invention provides pharmaceutical compositionscontaining a therapeutically effective amount of a compound of Formula(I) or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable excipient.

In a third aspect, this invention provides a method of treatment of adisease in a mammal treatable by administration of a CCR-3 receptorantagonist, comprising administration of a therapeutically effectiveamount of a compound of Formula (I) or a pharmaceutically acceptablesalt thereof. The disease states include respiratory diseases such asasthma.

In a fourth aspect, this invention provides processes disclosed hereinfor preparing compounds of Formula (I).

In a fifth aspect, this invention provides novel intermediates disclosedherein that are useful for preparing compounds of Formula (I).

In a sixth aspect, this invention provides a compound of Formula (I) ora pharmaceutically acceptable salt thereof for use in medical therapy ordiagnosis (e.g. for treating asthma).

In a seventh aspect, this invention provides the use of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof for themanufacture of a medicament useful for treating a disease in a mammaltreatable by administration of a CCR-3 receptor antagonist (e.g.asthma).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the meanings given below.

“Acyl” means a radical —C(O)R, where R is hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, phenyl or phenylalkyl wherein alkyl, cycloalkyl,cycloalkylalkyl, and phenylalkyl are as defined herein. Representativeexamples include, but are not limited to formyl, acetyl,cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl,and the like.

“Acylalkyl” means a radical -alkylene-C(O)R where R is hydrogen, alkyl,haloalkyl, cycloalkyl, cycloalkyl-alkyl, optionally substituted phenyl,benzyl, hydroxy, alkoxy, amino, monoalkylamino or dialkylamino.Representative examples include methylcarbonyl-methyl,2-(ethoxycarbonyl)ethyl, 2-(methoxycarbonyl)ethyl, 2-carboxyethyl andthe like.

“Acylamino” means a radical —NR′C(O)R, where R′0 is hydrogen or alkyl,and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl orphenylalkyl wherein alkyl, cycloalkyl, cycloalkylalkyl, and phenylalkylare as defined herein. Representative examples include, but are notlimited to formylamino, acetylamino, cylcohexylcarbonylamino,cyclohexylmethyl-carbonylamino, benzoylamino, benzylcarbonylamino, andthe like.

“Alkoxy” means a radical —OR where R is an alkyl as defined herein e.g.,methoxy, ethoxy, propoxy, butoxy and the like.

“Alkoxycarbonyl” means a radical —C(O)—R where R is alkoxy is as definedherein.

“Alkenyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one double bond, e.g., ethenyl,propenyl, and the like.

“Alkyl” means a linear saturated monovalent hydrocarbon radical of oneto six carbon atoms or a branched saturated monovalent hydrocarbonradical of three to six carbon atoms, e.g., methyl, ethyl, propyl,2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, and the like.

“Alkylamino” or “Monoalkylamino” means a radical —NHR where R representsan alkyl, cycloalkyl or cycloalkyl-alkyl group as defined herein.Representative examples include, but are not limited to methylamino,ethylamino, isopropylamino, cyclohexylamino, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms, e.g., methylene, ethylene,2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene,and the like.

“Alkynyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one triple bond, e.g., ethynyl,propynyl, and the like.

“Alkylsulfonyl” means a radical —S(O)₂R where R is an alkyl, cycloalkylor cycloalkylalkyl group as defined herein, e.g., methylsulfonyl,ethylsulfonyl, propylsulfonyl, butylsulfonyl, cyclohexylsulfonyl and thelike.

“Alkylsulfinyl” means a radical —S(O)R where R is an alkyl, cycloalkylor cycloalkylalkyl group as defined herein e.g., methylsulfinyl,ethylsulfinyl, propylsulfinyl, butylsulfinyl, cyclohexylsulfinyl and thelike.

“Alkylthio” means a radical —SR where R is an alkyl as defined abovee.g., methylthio, ethylthio, propylthio, butylthio, and the like.

“Aryl” means a monocyclic or bicyclic aromatic hydrocarbon radical whichis optionally substituted with one or more substituents, preferably one,two or three, substituents preferably selected from the group consistingof alkyl, haloalkyl, hydroxyalkyl, heteroalkyl, acyl, acylamino, amino,alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl,—SO₂NR′R″ (where R′ and R″ are independently hydrogen or alkyl), alkoxy,haloalkoxy, alkoxycarbonyl, carbamoyl, hydroxy, halo, nitro, cyano,mercapto, methylenedioxy or ethylenedioxy. More specifically the termaryl includes, but is not limited to, phenyl, chlorophenyl,fluorophenyl, methoxyphenyl, 1-naphthyl, 2-naphthyl, and the derivativesthereof.

“Arylene” means a divalent aryl group as defined above.

“Arylalkyl” refers to an alkyl radical as defined herein in which one ofthe hydrogen atoms of the alkyl group is replaced with an aryl group.Typical arylalkyl groups include, but are not limited to, benzyl,2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

“Aryloxy” means a radical —O—R where R is an aryl group as definedherein.

“Carbamoyl” means the radical —C(═O)NH₂

“Cycloalkyl” refers to a saturated monovalent cyclic hydrocarbon radicalof three to seven ring carbons e.g., cyclopropyl, cyclobutyl,cyclohexyl, 4-methylcyclohexyl, and the like.

“Cycloalkyl-alkyl” means a radical —R^(x)R^(y) where R^(x) is analkylene group and R^(y) is cycloalkyl group as defined herein, e.g.,cyclohexylmethyl, and the like.

“Dialkylamino” means a radical —NRR′ where R and R′ independentlyrepresent an alkyl, cycloalkyl, or cycloalkylalkyl group as definedherein. Representative examples include, but are not limited todimethylamino, methylethylamino, di(1-methylethyl)amino,(cyclohexyl)(methyl)amino, (cyclohexyl)(ethyl)amino,(cyclohexyl)(propyl)amino, (cyclohexylmethyl)(methyl)amino,(cyclohexylmethyl)(ethyl)amino, and the like.

“Halo” means fluoro, chloro, bromo, or iodo, preferably fluoro andchloro.

“Haloalkyl” means alkyl substituted with one or more same or differenthalo atoms, e.g., —CH₂Cl, —CF₃, —CH₂CF₃, —CH₂CCl₃, and the like.

“Heteroaryl” means a monocyclic or bicyclic radical of 5 to 12 ringatoms having at least one aromatic ring containing one, two, or threering heteroatoms selected from N, O, or S, the remaining ring atomsbeing C, with the understanding that the attachment point of theheteroaryl radical will be on an aromatic ring. The heteroaryl ring isoptionally substituted independently with one or more substituents,preferably one or two substituents, selected from alkyl, haloalkyl,hydroxyalkyl, heteroalkyl, acyl, acylamino, amino, alkylamino,dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, —SO₂NR′R″ (whereR′ and R″ are independently hydrogen or alkyl), alkoxy, haloalkoxy,alkoxycarbonyl, carbamoyl, hydroxy, halo, nitro, cyano, mercapto,methylenedioxy, ethylenedioxy or optionally substituted phenyl. Morespecifically the term heteroaryl includes, but is not limited to,pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl,imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyrimidinyl,5-(3,4-dimethoxyphenyl)-pyrimidin-2-yl,5-(4-methoxyphenyl)-pyrimidin-2-yl,5-(3,4-methylenedioxyphenyl)-pyrimidin-2-yl, benzofuranyl,tetrahydrobenzofuranyl, isobenzofuranyl, benzothiazolyl,benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyl,quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl,benzisoxazolyl or benzothienyl and derivatives thereof.

“Heteroarylene” means a divalent heteroaryl group as defined above.

“Heteroarylalkyl means an alkyl radical as defined herein in which oneof the hydrogen atoms of the alkyl group is replaced with a heteroarylgroup.

“Heteroalkyl” means an alkyl radical as defined herein wherein one, twoor three hydrogen atoms have been replaced with a substituentindependently selected from the group consisting of —OR^(a),—NR^(b)R^(c), and —S(O)_(n)R^(d) (where n is an integer from 0 to 2),with the understanding that the point of attachment of the heteroalkylradical is through a carbon atom, wherein R^(a) is hydrogen, acyl,alkyl, cycloalkyl, or cycloalkylalkyl; R^(b) and R^(c) are independentlyof each other hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl;when n is 0, R^(d) is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl,and when n is 1 or 2, R^(d) is alkyl, cycloalkyl, cycloalkylalkyl,amino, acylamino, monoalkylamino, or dialkylamino;. Representativeexamples include, but are not limited to, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxypropyl,1-hydroxymethylethyl, 3-hydroxybutyl, 2,3-dihydroxybutyl,2-hydroxy-1-methylpropyl, 2-aminoethyl, 3-aminopropyl,2-methylsulfonylethyl, aminosulfonylmethyl, aminosulfonylethyl,aminosulfonylpropyl, methylaminosulfonylmethyl,methylaminosulfonylethyl, methylaminosulfonylpropyl, and the like.

“Heterocyclyl” means a saturated or unsaturated non-aromatic cyclicradical of 3 to 8 ring atoms in which one or two ring atoms areheteroatoms selected from NR^(x) {wherein each R^(x) is independentlyhydrogen, alkyl, acyl, alkylsulfonyl, aminosulfonyl,(alkylamino)sulfonyl, (dialkylamino)sulfonyl, carbamoyl,(alkylamino)carbonyl, (dialkylamino)carbonyl, (carbamoyl)alkyl,(alkylamino)carbonylalkyl, or dialkylaminocarbonylalkyl}, O, or S(O)_(n)(where n is an integer from 0 to 2), the remaining ring atoms being C.The heterocyclyl ring may be optionally substituted independently withone, two, or three substituents selected from alkyl, haloalkyl,heteroalkyl, halo, nitro, cyanoalkyl, hydroxy, alkoxy, amino,monoalkylamino, dialkylamino, aralkyl, —(X)_(n)—C(O)R (where X is O orNR′, n is 0 or 1, R is hydrogen, alkyl, haloalkyl, hydroxy, alkoxy,amino, monoalkylamino, dialkylamino or optionally substituted phenyl,and R′ is hydrogen or alkyl), -alkylene-C(O)R (where R is hydrogen,alkyl, haloalkyl, hydroxy, alkoxy, amino, monoalkylamino, dialkylaminoor optionally substituted phenyl) or —S(O)_(n)R^(d) (where n is aninteger from 0 to 2, and R^(d) is hydrogen (provided that n is 0),alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, amino, monoalkylamino,dialkylamino, or hydroxyalkyl). More specifically the term heterocyclylincludes, but is not limited to, tetrahydropyranyl, piperidino,N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl,3-pyrrolidino, morpholino, thiomorpholino, thiomorpholino-1-oxide,thiomorpholino-1,1-dioxide, tetrahydrothiophenyl-S,S-dioxide,pyrrolinyl, imidazolinyl, and the derivatives thereof.

“Hydroxyalkyl” means an alkyl radical as defined herein, substitutedwith one or more, preferably one, two or three hydroxy groups, providedthat the same carbon atom does not carry more than one hydroxy group.Representative examples include, but are not limited to, 2-hydroxyethyl,2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl,2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl,2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyland 2-(hydroxymethyl)-3-hydroxypropyl, preferably 2-hydroxyethyl,2,3-dihydroxypropyl and 1-(hydroxymethyl)-1-hydroxyethyl. Accordingly,as used herein, the term “hydroxyalkyl” is used to define a subset ofheteroalkyl groups.

“Leaving group” has the meaning conventionally associated with it insynthetic organic chemistry, i.e., an atom or a group capable of beingdisplaced by a nucleophile and includes halo (such as chloro, bromo, andiodo), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g.,acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy,trifluoromethanesulfonyloxy, aryloxy (e.g., 2,4-dinitrophenoxy),methoxy, N,O-dimethylhydroxylamino, and the like.

“Optionally substituted phenyl” means a phenyl group which is optionallysubstituted with one or more substituents, preferably one, two or three,substituents preferably selected from the group consisting of alkyl,haloalkyl, hydroxyalkyl, heteroalkyl, acyl, acylamino, amino,alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl,—SO₂NR′R″ (where R′ and R″ are independently hydrogen or alkyl), alkoxy,haloalkoxy, alkoxycarbonyl, carbamoyl, hydroxy, halo, nitro, cyano,mercapto, methylenedioxy or ethylenedioxy. More specifically the termincludes, but is not limited to, phenyl, chlorophenyl, fluorophenyl,bromophenyl, methylphenyl, ethylphenyl, methoxyphenyl, cyanophenyl,4-nitrophenyl, 4-trifluoromethylphenyl, 4-chlorophenyl,3,4-difluorophenyl, 2,3-dichlorophenyl, 3-methyl-4-nitrophenyl,3-chloro-4-methylphenyl, 3-chloro-4-fluorophenyl or 3,4-dichlorophenyland the derivatives thereof.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “aryl group optionally mono- ordi-substituted with an alkyl group” means that the alkyl may but neednot be present, and the description includes situations where the arylgroup is mono- or disubstituted with an alkyl group and situations wherethe aryl group is not substituted with the alkyl group.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

“Phenylalkyl” refers to an alkyl radical as defined herein in which oneof the hydrogen atoms of the alkyl radical has been replaced by anoptionally substituted phenyl.

“Protecting group” refers to a grouping of atoms that when attached to areactive group in a molecule masks, reduces or prevents that reactivity.Examples of protecting groups can be found in T. W. Green and P. G.Futs, Protective Groups in Organic Chemistry, (Wiley, 2^(nd) ed. 1991)and Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996). Representativeamino protecting groups include, formyl, acetyl, trifluoroacetyl,benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethylsilyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl andsubstituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC),and the like. Representative hydroxy protecting groups include thosewhere the hydroxy group is either acylated or alkylated such as benzyl,and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers,trialkylsilyl ethers and allyl ethers.

“Treating” or “treatment” of a disease includes: (1) preventing thedisease, i.e., causing the clinical symptoms of the disease not todevelop in a mammal that may be exposed to or predisposed to the diseasebut does not yet experience or display symptoms of the disease; (2)inhibiting the disease, i.e., arresting or reducing the development ofthe disease or its clinical symptoms; or (3) relieving the disease,i.e., causing regression of the disease or its clinical symptoms.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

Compounds that have the same molecular Formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers”. When a compound has an asymmetric center, for example, ifa carbon atom is bonded to four different groups, a pair of enantiomersis possible. An enantiomer can be characterized by the absoluteconfiguration of its asymmetric center and is described by the R- andS-sequencing rules of Cahn and Prelog, or by the manner in which themolecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see discussion in Chapter 4 of “Advanced OrganicChemistry”, 4th edition J. March, John Wiley and Sons, New York, 1992).

In general, the nomenclature used in this Application is based onAUTONOM™, a Beilstein Institute computerized system for the generationof IUPAC systematic nomenclature. For example, a compound of Formula (I)wherein R₁ is methylene; R₂ is 3,4-dichlorophenyl; L is C(═O); A isphenyl; R₃ is hydrogen; and R₄ is 3,4-difluorophenyl; and X is absent(Example 1), is namedN-{2-[4-((3E,5E)-4,5-Dichloro-2-methylene-hepta-3,5-dienyl)-piperazin-1-yl]-phenyl}-3,4-difluoro-benzamide.

Resentative compounds of Formula (I) are shown in the following table.

Cpd. M.P. No. Structure (° C.) Example 1

5 2

1 3

4

4 5

6 6

3 7

190.8-204.3 2 8

7 9

PREFERRED EMBODIMENTS

While the broadest definition of this invention is set forth in theSummary of the Invention, certain compounds of Formula (I) arepreferred.

A preferred compound of the invention is a compound of Formula (I)wherein R¹ is methylene.

Another preferred compound of the invention are compounds of Formula (I)wherein ring A is cyclopentyl. Compounds where ring A is cyclopentyl arebind unexpectedly potently to the CCR-3 receptor. Other preferredcompounds of the invention are compounds of Formula (I) wherein ring Ais heterocyclyl (particularly tetrahydropyranyl,S,S-dioxo-tetrahydothiophenyl, tetrahydrothiophenyl or pyrrolidinyl) orcompounds of Formula (I) wherein ring A is phenyl.

A preferred compound of the invention is a compound of Formula (I)wherein R² is phenyl ring substituted with one, or two substituentsselected from alkyl, alkoxy, haloalkyl, halo, cyano or nitro; preferablymethyl, ethyl, methoxy, trifluoromethyl, chloro, fluoro or bromo; mostpreferably 4-nitrophenyl, 4-trifluoromethylphenyl, 4-chlorophenyl,3,4-difluorophenyl, 2,3-dichlorophenyl, 3-methyl-4-nitrophenyl,3-chloro-4-methylphenyl, 3-chloro-4-fluorophenyl or 3,4-dichlorophenyl.Particularly preferred are 4-chlorophenyl or 3,4-dichlorophenyl. Suchsubstituted compounds show particularly good binding to the CCR-3receptor compared to their unsubstituted phenyl analogues.

A preferred compound of the invention is a compound of Formula (I)wherein R³ is hydrogen or methyl, preferably hydrogen.

A preferred compound of the invention is a compound of Formula (I)wherein L is —C(═O)—, —SO₂—, —C(═O)N(R_(a))—, —C(═S)N(R_(a))—, or—C(═O)O—. More preferred are compounds where L is —C(═O)—,—C(═O)N(R_(a))—, most preferably —C(═O)N(R_(a))—. In the preceding R_(a)is preferably hydrogen or methyl, most preferably hydrogen.

A preferred compound of the invention is a compound of Formula (I)wherein X is absent, methylene, 1,2-ethanediyl, 1,3-propanediyl, or1,4-butanediyl.

A preferred compound of the invention is a compound of Formula (I)wherein R⁴ is optionally substituted phenyl, optionally substitutedheteroaryl wherein the heteroaryl ring is indolyl, thienyl, quinolinylor 1,8-naphthyridinyl. Preferably R⁴ is selected from3,4-dichlorophenyl, 3,4,5-trimethoxyphenyl, 4-methanesulfonyl-phenyl,3-methanesulfonylphenyl, 4-methoxynaphthalen-2-yl,5-(3,4-dimethoxyphenyl)pyrimidin-2-yl, phenyl, 3-fluorophenyl,4-ethylphenyl, 3-methoxyphenyl, 2,4-difluorophenyl,3-trifluoromethylphenyl, 4-methylphenyl, 4-fluorophenyl, 2-fluorophenyl,4-carboxamidophenyl, 4-acetylphenyl, 4-nitrophenyl, 2-methylphenyl,2-chloro-4-fluorophenyl, 3,4-dimethoxyphenyl, 2,5-dimethoxyphenyl,2,3-dichlorophenyl, 2,4-dichlorophenyl, 4-bromophenyl,4-chloro-3-nitrophenyl, 2-nitrophenyl, 2-nitro-4-trifluoromethylphenyl,4-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 3-methylphenyl,2-trifluoromethylphenyl, 2-methoxyphenyl, 3-bromophenyl,4-trifluoromethylphenyl, 3-trifluoromethyl, 3,5-bis-trifluoromethyl,4-tert-butylphenyl, 4-ethoxyphenyl, 3-cyanophenyl, 4-cyanophenyl,4-methoxyphenyl, 3-nitrophenyl, 3,5-dimethoxyphenyl, 4-iodophenyl,4-isopropylphenyl, 3-methoxycarbonylphenyl 3-acetylphenyl,2-methylphenyl, indol-2-yl, 5-methoxyindol-2-yl, 5-chloroindol-2-yl,2-methoxycarbonylphenyl, 3,5-dichlorophenyl, 1-naphthyl,3-chloro-2-methylphenyl, 2,5-dimethylphenyl, 2-thienyl, 3-ethoxyphenyl,3-isoquinolyl, 2-methylquinolin-6-yl, 3-methylaminophenyl, 3-quinolyl,2-quinolyl, 5-hydroxynaphthalen-2-yl, 8-hydroxyquinolin-2-yl,5,7-dimethyl-[1,8]naphthyridin-2-yl, 6-quinolyl, 3-(acetylamino)phenylor 2,3,4-trimethoxyphenyl.

Also preferred are compounds where X is —CH₂S—, —CH₂O—, —CH₂CH₂— and R⁴is heteroaryl, preferably optionally substituted pyrimidinyl, pyrazolylor thienyl. Particularly preferred are compounds where X is —CH₂S— andR⁴ is 5-(3,4-dimethoxyphenyl)-pyrimidin-2-yl,5-(3,4-methylenedioxy)-pyrimidin-2-yl, 5-(4-methoxyphenyl)pyrimidin-2-yl

A specific compound of Formula (I) is a compound of Formula (II):

wherein R¹-R⁴, A, L, and X have any of the values described herein.

A specific compound of Formula (I) is a compound of Formula (III):

wherein R¹-R⁴, A, L, and X have any of the values described herein.

A specific compound of formula (I) is a compound of formula (IV):

wherein R³,R⁴, A, L, and X have any of the values described herein.

A specific compound of Formula (I) is a compound of formula (V):

wherein X and R⁴ have any of the values defined herein.

A specific compound of Formula (I) is a compound of formula (VI):

wherein X and R⁴ have any of the values defined herein.

A specific compound of Formula (I) is a compound of formula (VII):

wherein X and R⁴ have any of the values defined herein.

A specific compound of Formula (I) is a compound of formula (VIII):

wherein X and R⁴ have any of the values defined herein; and R^(x) ishydrogen, alkyl, acyl, alkylsulfonyl, aminosulfonyl,(alkylamino)sulfonyl, (dialkylamino)sulfonyl, carbamoyl,(alkylamino)carbonyl, (dialkylamino)carbonyl, (carbamoyl)alkyl,(alkylamino)carbonylalkyl, or dialkylaminocarbonylalkyl.

A specific compound of Formula (I) is a compound of formula (IX):

wherein X and R⁴ have any of the values defined herein.

A specific compound of Formula (I) is a compound of formula (X):

wherein X and R⁴ have any of the values defined herein.

A particularly preferred compound of the invention is:

N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-2-thiophen-2-yl-acetamide;

N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-3,4-difluoro-benzamide;

N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-cyclohexyl}-4-methyl-benzamide;

1-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-3-(3-methoxy-phenyl)-urea;

4-Chloro-N-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-benzamide;

N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-4-methyl-benzenesulfonamide;

1-{4-[4-(4-Chloro-benzyl)-piperazin-1-yl]-tetrahydro-furan-3-yl}-3-(3,4,5-trimethoxy-phenyl)-urea.

or a pharmaceutically acceptable salt thereof.

General Utility

The compounds of the invention are CCR-3 receptor antagonists andinhibit eosinophil recruitment by CCR-3 chemokines such as RANTES,eotaxin, MCP-2, MCP-3 and MCP-4. Compounds of this invention andcompositions containing them are useful in the treatment ofeosiniphil-induced diseases such as inflammatory or allergic diseasesand including respiratory allergic diseases such as asthma, allergicrhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis,eosinophilic pneumonias (e.g., chronic eosinophilic pneumonia);inflammatory bowel diseases (e.g., Crohn's disease and ulcerativecolitis); and psoriasis and inflammatory dermatoses such as dermatitisand eczema.

Testing The CCR-3 antagonistic activity of the compounds of thisinvention can be measured by in vitro assays such as ligand binding andchemotaxis assays as described in more detail in Examples 9, 10, and 11.In vivo activity can be assayed in the Ovalbumin induced Asthma inBalb/c Mice Model as described in more detail in Example 12.Administration and Pharmaceutical Composition

In general, the compounds of this invention can be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the compound of this invention, i.e., the active ingredient,will depend upon numerous factors such as the severity of the disease tobe treated, the age and relative health of the subject, the potency ofthe compound used, the route and form of administration, and otherfactors.

Therapeutically effective amounts of compounds of Formula (I) may rangefrom approximately 0.01-20 mg per kilogram body weight of the recipientper day; preferably about 0.1-10 mg/kg/day. Thus, for administration toa 70 kg person, the dosage range would most preferably be about 7 mg to0.7 g per day.

In general, compounds of this invention will be administered aspharmaceutical compositions by any one of the following routes: oral,transdermal, inhalation (e.g., intranasal or oral inhalation) orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration. A preferred manner of administration is oral using aconvenient daily dosage regimen which can be adjusted according to thedegree of affliction. Compositions can take the form of tablets, pills,capsules, semisolids, powders, sustained release Formulations,solutions, suspensions, liposomes, elixirs, or any other appropriatecompositions. Another preferred manner for administering compounds ofthis invention is inhalation. This is an effective means for deliveringa therapeutic agent directly to the respiratory tract for the treatmentof diseases such as asthma and other similar or related respiratorytract disorders (see U.S. Pat. No. 5,607,915).

The choice of Formulation depends on various factors such as the mode ofdrug administration and the bioavailability of the drug substance. Fordelivery via inhalation the compound can be Formulated as liquidsolutions or suspensions, aerosol propellants or dry powder and loadedinto a suitable dispenser for administration. There are three types ofpharmaceutical inhalation devices—nebulizer inhalers, metered-doseinhalers (MDI) and dry powder inhalers (DPI). Nebulizer devices producea stream of high velocity air that causes the therapeutic agents (whichhas been Formulated in a liquid form) to spray as a mist which iscarried into the patient's respiratory tract. MDI's typically have theFormulation packaged with a compressed gas. Upon actuation, the devicedischarges a measured amount of therapeutic agent by compressed gas,thus affording a reliable method of administering a set amount of agent.DPI's administer therapeutic agents in the form of a free flowing powderthat can be dispersed in the patient's inspiratory air-stream duringbreathing by the device. In order to achieve a free flowing powder, thetherapeutic agent is formulated with an excipient, such as lactose. Ameasured amount of the therapeutic is stored in a capsule form and isdispensed to the patient with each actuation. Recently, pharmaceuticalFormulations have been developed especially for drugs that show poorbioavailability based upon the principle that bioavailability can beincreased by increasing the surface area i.e., decreasing particle size.For example, U.S. Pat. No. 4,107,288 describes a pharmaceuticalFormulation having particles in the size range from 10 to 1,000 nm inwhich the active material is supported on a crosslinked matrix ofmacromolecules. U.S. Pat. No. 5,145,684 describes the production of apharmaceutical formulation in which the drug substance is pulverized tonanoparticles (average particle size of 400 nm) in the presence of asurface modifier and then dispersed in a liquid medium to give apharmaceutical formulation that exhibits remarkably highbioavailability.

The compositions are comprised of in general, a compound of Formula (I)in combination with at least one pharmaceutically acceptable excipient.Acceptable excipients are non-toxic, aid administration, and do notadversely affect the therapeutic benefit of the compound of Formula (I).Such excipient may be any solid, liquid, semi-solid or, in the case ofan aerosol composition, gaseous excipient that is generally available toone of skill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc.

For liposomal formulations of the drug for parenteral or oral deliverythe drug and the lipids are dissolved in a suitable organic solvent e.g.tert-butanol, cyclohexane (1% ethanol). The solution is lyopholized andthe lipid mixture is suspended in an aqueous buffer and allowed to forma liposome. If necessary, the liposome size can be reduced bysonification. (see, Frank Szoka, Jr. and Demetrios Papahadjopoulos,“Comparative Properties and Methods of Preparation of Lipid Vesicles(Liposomes)”, Ann. Rev. Biophys. Bioeng., 9:467-508 (1980), and D. D.Lasic, “Novel Applications of Liposomes”, Trends in Biotech.,16:467-608, (1998)).

Other suitable pharmaceutical excipients and their formulations aredescribed in Remington's Pharmaceutical Sciences, edited by E. W. Martin(Mack Publishing Company, 18th ed., 1990).

The level of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent (wt %) basis, from about 0.01-99.99 wt% of a compound of Formula (I) based on the total formulation, with thebalance being one or more suitable pharmaceutical excipients.Preferably, the compound is present at a level of about 1-80 wt %.Representative pharmaceutical formulations containing a compound ofFormula (I) are described in Example 8.

GENERAL SYNTHETIC SCHEME

Synthesis of Compounds of Formula (I)

The compounds of the present invention can be prepared in a number ofways known to one skilled in the art. Preferred methods include, but arenot limited to, the general synthetic procedures described below.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCo., (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), EnikaChemie, or, Sigma (St. Louis, Mo., USA), Maybridge (Dist: RyanScientific, P.O. Box 6496, Columbia, S.C. 92960), Bionet Research Ltd.,(Cornwall PL32 9QZ, UK), Menai Organics Ltd., (Gwynedd, N. Wales, UK),Butt Park Ltd., (Dist. Interchim, Montlucon Cedex, France) or areprepared by methods known to those skilled in the art followingprocedures set forth in references such as Fieser and Fieser's Reagentsfor Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd'sChemistry of Carbon Compounds, Volumes 1-5 and Supplementals (ElsevierScience Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wileyand Sons, 1991), March's Advanced Organic Chemistry, (John Wiley andSons, 1992), and Larock's Comprehensive Organic Transformations (VCHPublishers Inc., 1989). These schemes are merely illustrative of somemethods by which the compounds of this invention can be synthesized, andvarious modifications to these schemes can be made and will be suggestedto one skilled in the art having referred to this disclosure.

The starting materials and the intermediates of the reaction may beisolated and purified if desired using conventional techniques,including but not limited to filtration, distillation, crystallization,chromatography, and the like. Such materials may be characterized usingconventional means, including physical constants and spectral data.

Compounds of Formula (I) are generally prepared from the precursor amineof Formula (Ia) as shown below.

Preparation of compounds of Formula (Ia) and their conversion tocompounds of Formula I is illustrated in the following Schemes 1-8.

Schemes 1-5 show methods of preparing compounds of Formula Ia havingdifferent rings A. Specific exemplification is provided for R¹—R² being4-chlorobenzyl in Preparations 1-6. Preparation of analogous compoundswhere R¹ and R² vary within the full scope of the Summary of theInvention may be readily prepared by one of skill in the art in light ofthis specification and incorporated references.

1.3.1 General Procedure A: (Amine Alkylation with Epoxides)

A 0.5-1.5 M solution of the amine, R₂NH (1 equiv), and the specifiedepoxide, 3a (1.1-10 equiv) in EtOH is stirred at 80-95° C. for 2-4.5 d,allowed to cool to room temperature, and concentrated. The crude aminoalcohol is purified by chromatography or recrystallization.

1.3.2 General Procedure B: (Amine Formation Using MethanesulfonylChloride and Ammonium Hydroxide)

A 0.2-0.3 M solution of the amino alcohol (1 equiv) in CH₂Cl₂ at 0° C.is treated successively with Et₃N (2 equiv) and MeSO₂Cl (2 equiv),stirred at 0° C. for 1-2 hours, and partitioned between CH₂Cl₂ and10-15% NH₄OH. The aqueous phase is extracted with CH₂Cl₂ and theextracts are dried and concentrated. A 0.13M solution of the residue in2.5:1 dioxane: 28-30 wt % NH₄OH is stirred at 70-80° C. 2.5-18 hours,allowed to cool to room temperature, and concentrated. The residue ispartitioned between EtOAc and 1 N NaOH, the aqueous phase is extractedwith EtOAc, and the extracts are washed with brine, dried andconcentrated. The crude product is purified by chromatography or usedwithout further purification.

Schemes 6 and 7 show preparation of compounds of Formula Ia where ring Ais substituted. Scheme 6 shows preparation of compounds of Formula Iawith a substituted cyclopentyl ring A. Scheme 7 shows preparation ofcompounds of Formula Ia with a substituted pyrrolidine ring A bytreatment of the unsubstituted pyrrolidine 7a (R=H) with the appropriatereagent to produce the substituted pyrrolidine 7b.

Schemes 8 and 9 show methods of converting compounds of Formula (Ia) tocompounds of Formula (I) where L and A are varied.

1.8.1 General Procedure C: (Urea Formation Using Isocyanates)

A 0.1-0.6 M solution of the amine (1 equiv) in CH₂Cl₂ or CH₂Cl₂ and DMFat 0-20° C. is treated with the specified isocyanate (1.1-2 equiv),stirred for 0.5-1.5 hours, and partitioned between CH₂Cl₂ and saturatedNaHCO₃. The aqueous phase is extracted with CH₂Cl₂ and the extracts aredried and concentrated. The crude urea is purified by columnchromatography or preparative TLC or used in the next step withoutfurther purification.

1.8.2 General Procedure D: (Urea Formation Using Isocyanates)

A 0.1-0.6 M solution of the amine (1 equiv) in CH₂Cl₂ or CH₂Cl₂ and DMFat 0-20° C. is treated with the specified isocyanate (1.1-2 equiv),stirred for 0.5-1.5 hours, and partitioned between CH₂Cl₂ and saturatedNaHCO₃. The aqueous phase is extracted with CH₂Cl₂ and the extracts aredried and concentrated. The crude urea is purified by columnchromatography or preparative TLC or used in the next step withoutfurther purification. A solution of the free base in CH₂Cl₂ is treatedwith 1 N HCl in Et₂O and concentrated to give the hydrochloride salt.

1.8.3 General Procedure E: (Amide Formation Using 1-Hydroxybenzotriazoleand 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride)

A 0.1-0.4 M solution of the amine (1 equiv) and the specified carboxylicacid (1.2-1.5 equiv) in CH₂Cl₂ at 0° C. is treated successively with1-hydroxybenzotriazole hydrate (HOBt) (0.2-0.5 equiv) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DEC)(1.3-2 equiv), stirred at 0-20° C. for 2-72 hours, and partitionedbetween CH₂Cl₂ and NaHCO₃. The aqueous phase is extracted with CH₂Cl₂and the extracts are dried and concentrated. The crude amide is purifiedby column chromatography and/or preparative TLC.

1.8.4 General Procedure F: (Amide Formation Using 1-Hydroxybenzotriazoleand 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride)

A 0.1-0.4 M solution of the amine (1 equiv) and the specified carboxylicacid (1.2-1.5 equiv) in CH₂Cl₂ at 0° C. is treated successively with1-hydroxybenzotriazole hydrate (HOBt) (0.2-0.5 equiv) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (DEC)(1.3-2 equiv), stirred at 0-20° C. for 2-72 hours, and partitionedbetween CH₂Cl₂ and saturated NaHCO₃. The aqueous phase is extracted withCH₂Cl₂ and the extracts are dried and concentrated. The crude amide ispurified by column chromatography and/or preparative TLC. A solution ofthe free base in CH₂Cl₂ is treated with 1 N HCI in Et₂O and concentratedto provide the hydrochloride salt.

2.1.1 General Procedure G (Parallel Synthesis of Sulfonamides)

A mixture of the requisite amine Ia (1 equiv), the appropriate sulfonylchloride (1.5 equiv), and Amberlite IRA67 (2 equiv) in CH₂Cl₂ (2 mL) wasrotated overnight. The mixture was treated with PS-trisamine (1.2 equiv)(Argonaut Technologies Inc., San Carlos, Calif., USA) and rotatedovernight. The solid was collected by filtration and washed with CH₂Cl₂,MeOH, and CH₂Cl₂. The filtrate was concentrated to give the product.

2.1.2 General Procedure H (Parallel Synthesis of Amides from AcidChlorides)

A mixture of the requisite amine Ia (1 equiv), the appropriate acidchloride (1.5 equiv), and Amberlite IRA67 (2 equiv) in CH₂Cl₂ (2 mL) wasrotated overnight. The mixture was treated with PS-trisamine (1.2 equiv)and MP-carbonate (2 equiv) (Argonaut Technologies, San Carlos, Calif.)and rotated overnight. The solid was collected by filtration and washedwith CH₂Cl₂, MeOH, and CH₂Cl₂. The filtrate was concentrated to give theproduct.

2.1.3 General Procedure I (Parallel Synthesis of Amides from CarboxylicAcids)

A mixture of the requisite amine Ia (1 equiv), the appropriatecarboxylic acid (1.5 equiv), and PS-carobodiimide (2 equiv) (ArgonautTechnologies Inc., San Carlos, Calif., USA) in CH₂Cl₂ (2 mL) was rotatedovernight. The mixture was treated with MP-carbonate (2 equiv) androtated overnight. The solid was collected by filtration and washed withCH₂Cl₂, MeOH, and CH₂Cl₂. The filtrate was concentrated to give theproduct.

2.1.4 General Procedure J (Parallel Synthesis of Ureas from Isocyanatesand Purification by Parallel Chromatography)

A mixture of the requisite amine Ia (1 equiv) and the appropriateisocyanate (1.2 equiv) in CH₂Cl₂ (2 mL) was stirred overnight. Themixture was concentrated to give the crude product, which was purifiedby parallel chromatography using a step gradient (2.5% MeOH/CH₂Cl₂, 10%MeOH/CH₂Cl₂).

2.1.5 General Procedure K (Parallel Synthesis of Ureas from Isocyanatesand Purification by Catch and Release Scavenger)

A mixture of the requisite amine Ia (1 equiv) and the appropriateisocyanate (1.2 equiv) in CH₂Cl₂ (2 mL) was stirred overnight. Themixture was treated with MP-TsOH and rotated for 3 h. The solid wascollected by filtration and washed with CH₂Cl₂, MeOH, and CH₂Cl₂. Thesolid was rotated with 2 M NH₃ in MeOH for 2 h. The solid was collectedby filtration and washed with CH₂Cl₂, MeOH, and CH₂Cl₂. The filtrate wasconcentrated to give the purified product.

2.1.6 General Procedure L (Parallel Synthesis of Ureas from Anilinesusing Phoxime Resin)

A mixture of the appropriate aniline (3 equiv) and Phoxime resin (1equiv) in CH₂Cl₂ (2 mL) was rotated for 3 h. If the aniline had notdissolved, triethylarmine (3.5 equiv) was added. The mixture was rotatedovernight. The solid was collected by filtration and washed with CH₂Cl₂,MeOH, CH₂Cl₂, MeOH, and CH₂Cl₂. A mixture of the solid and the requisiteamine Ia (1.1 equiv) in CH₂Cl₂ (0.5 mL) and toluene (1.5 mL) were heatedat 80° C. with shaking overnight and allowed to cool to roomtemperature. The solid was collected by filtration and washed withCH₂Cl₂, MeOH, and CH₂Cl₂. The filtrate was concentrated to give theproduct.

2.1.7 General Procedure M (Parallel Synthesis of Ureas from Anilinesusing Triphosgene)

A mixture of the appropriate aniline (1.2 equiv), triphosgene (0.4equiv), and triethylamine (1.4 equiv) in CH₂Cl₂ was heated at 35° C. for1 h. After cooling to room temperature, the requisite amine Ia (1 equiv)was added. The mixture was stirred overnight, washed with H₂O and brine,passed through Na₂SO₄, and concentrated to give crude product which waspurified by parallel chromatography.

2.1.8 General Procedure N (Parallel Synthesis of Thioureas fromThioisocyanates)

A mixture of the requisite amine Ia (1 equiv) and the appropriatethioisocyanate (1.2 equiv) in CH₂Cl₂ (2 mL) was stirred overnight. Themixture was treated with MP-TsOH and rotated for 3 h. The solid wascollected by filtration and washed with CH₂Cl₂, MeOH, and CH₂Cl₂. Thesolid was rotated with 2 M NH₃ in MeOH for 2 h. The solid was collectedby filtration and washed with CH₂Cl₂, MeOH, and CH₂Cl₂. The filtrate wasconcentrated to give the purified product.

2.1.9 General Procedure O (Parallel Synthesis of Carbamates)

A mixture of the requisite amine Ia (1 equiv) and the appropriatesuccinimide (1.5 equiv) in CH₂Cl₂ (2 mL) was stirred overnight. If thereaction was not complete, it was heated at 38° C. for 1 hour. Themixture was washed with H₂O and brine, passed through Na₂SO₄ andconcentrated to give the crude product which was purified via parallelpurification (step gradient 5% MeOH/CH₂Cl₂, 10% MeOH/CH₂Cl₂).

3. EXPERIMENTAL SECTION

General

Unless otherwise noted, all non-aqueous reactions were run under anitrogen atmosphere and Na₂SO₄ was used to dry all organic layers.Purifications were typically carried out by flash chromatography onsilica gel (230-400 mesh) or preparative TLC on Uniplate Silica Gel GFPLC Plates (20×20 cm, 1000 microns) from Analtech, Inc., Newark, Del.Alumina used was basic with 6 wt % H₂O (Brockmann III). Melting pointstaken in capillary tubes are uncorrected. IR spectra were determined inKBr. NMR spectra were run in CDCl3, unless otherwise indicated. ¹H NMRspectra were recorded on 300 MHz instruments and ¹³C NMR spectra wererecorded at 75.5 MHz. Mass spectral analyses were accomplished usingelectrospray ionization. Analytical reverse-phase HPLC was performed onShimadzu system equipped with a diode array spectrometer (range 190-300nm; Hewlett Packard). The stationary phase was a Zorbax SB-Phenyl RapidResolution column (4.6 mm×50 mm; Hewlett Packard), mobile phase A was0.1% trifluoroacetic acid, and mobile phase B was CH₃CN. A flow rate of2.5 mL/min with a linear gradient of 20-55% B in 5 min and then 55-20% Bin 5 min was employed. Other physical and analytical data were obtainedby the physical and analytical chemistry group at Roche Bioscience. Allparallel synthesis reactions were run in sealed tubes that were ventedprior to being rotated overnight. Amberlite IRA67 (Aldrich Chemical Co.,Milwaukee, Wis., USA)was washed consecutively with CH₂Cl₂, MeOH, CH₂Cl₂,MeOH, CH₂Cl₂ and then dried under vacuum prior to use. All productsderived from parallel synthesis reactions were characterized viaHPLC-MS.

4. EXAMPLES

5. The following Preparations and Examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

6. The following General Procedures (A-O) are useful for preparingcompounds of the invention. These General Procedures are referred to inthe following Examples.

6.1.1.1 Preparation 1

1-(3,4-dichlorobenzyl)piperazine and 1-(4-chlorobenzyl)piperazine

Step 1

3,4-Dichlorobenzyl bromide (35.20 g, 150 mmol) was added to a solutionof N-(tert-butoxycarbonyl)piperazine (24.84 g, 130 mmol) andtriethylamine (20.91 ml, 150 mmol) in chloroform (100 ml) over 30 min.After 1 h, the reaction mixture was diluted with ethyl acetate and theproduct was precipitated out as the hydrochloride salt by adding 1Naqueous hydrogen chloride solution. The solid product was filtered,washed with water and then resuspended in ethyl acetate. Two equivalentsof 1N aqueous sodium hydroxide solution was added and the free amine wasextracted into ethyl acetate. The organic layer was separated, driedover magnesium sulfate, filtered and concentrated to provide1-(tert-butoxycarbonyl)-4-(3,4-dichlorobenzyl)piperazine (45 g).

Step 2

Trifluoroacetic acid (75 ml, 974 mmol) was added to a solution1-(tert-butoxycarbonyl)-4-(3,4-dichlorobenzyl)piperazine (45 g, 130mmol) in chloroform (75 ml). The reaction mixture was stirred for 1 h atroom temperature and then made basic with a sodium hydroxide solution.The product was extracted into ethyl acetate and the organic layer waswashed with sodium bicarbonate solution, dried over magnesium sulfateand concentrated in vacuo to give 1-(3,4-dichlorobenzyl)piperazine (35.8g) as a solid.

1-(4-chlorobenzyl)piperazine was obtained by replacing3,4-dichlorobenzylbromide in Step 1 with 4-chlorobenzylbromide.

6.1.2 Example 1

Preparation ofN-{2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenyl}-3,4-difluoro-benzamide

2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine (45 mg) wasdissolved in a solution of triethylamine in dichloromethane (3 mL, 0.29M in triethylamine). This solution was added to a test tube containing3,4-difluorobenzoyl chloride (65 mg). After standing at ambienttemperature for several days, the reaction was directly passed through asmall pad of silica and eluted with 10% ethyl acetate in hexanes, thenwith 50% ethyl acetate in hexanes. Solvent removal left 49.8 mg of theproduct: MS m/z 475 (M)⁺. R00114-9313

6.1.3 Step A: Preparation of1-(3,4-dichlorobenzyl)-4-(2-nitrophenyl)-piperazine

A mixture of 1-(2-nitro-phenyl)-piperazine (4.53 g) (commercially fromEmkachem) and 3,4-dichlorobenzyl bromide (4.6 g) in 100 mL ofdichloromethane was treated with triethylamine (3.4 mL). The reactionwas stirred at ambient temperature under inert atmosphere overnight.After solvent removal, the crude reaction mixture was subjected directlyto flash chromatography using silica. Elution with hexanes, then 20%ethyl acetate in hexanes, and finally 33% ethyl acetate in hexanesfurnished the product as an oil (6.4 g), which was carried on as is.

6.1.4 Step B: Preparation of2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine

A solution of 1-(3,4-dichlorobenzyl)-4-(2-nitro-phenyl)-piperazine (1.19g) in 50 mL of 4/1 ethyl acetate/methanol was treated with 5% palladiumon carbon (55 mg) and placed under a hydrogen atmosphere. After stirringat ambient temperature overnight, the reaction was filtered using a padof silica and the pad was washed with 17% ethyl acetate in hexanes, then33% ethyl acetate in hexanes. Removal of solvent afforded 0.75 g of theproduct as a solid.

7. 7.1.1 Example 2

Preparation of(±)-trans-1-{4-[4-(4-chlorobenzyl)-piperazin-1-yl]-tetrahydrofuran-3-yl}-3-(3,4,5-trimethoxyphenyl)ureadihydrochloride

Following General Procedure D,(±)-trans-4-[4-(4-chlorobenzyl)piperazin-1-yl]-tetrahydrofuran-3-ylamine(120 mg, 0.41 mmol) and 5-isocyanato-1,2,3-trimethoxybenzene (102 mg,0.50 mol) were coupled in CH₂Cl₂ (2 mL) at 0° C. for 1 h. Purificationof the crude product by preparative TLC with 100% EtOAc followed by asubsequent preparative TLC with 10:0.95:0.05 CH₂Cl₂:MeOH:NH₄OH gave thefree base (160 mg, 0.32 mmol) as a white solid. A solution of the freebase in CH₂Cl₂ was treated with 1 N HCl in Et₂O (0.8 mL, 0.8 mmol) andconcentrated to give the product (180 mg, 77%) as a pale tan solid: mp190.8-204.3° C.; MS m/z 505 (M+H)⁺. R0331-0138

7.1.2 Step A: Preparation of(±)-trans-4-[4-(4-chlorobenzyl)-piperazin-1-yl]-tetrahydrofuran-3-ol

Following General Procedure A, 1-(4-chlorobenzyl)-piperazine (500 mg,2.37 mmol) was alkylated with 3,6-dioxa-bicyclo[3.1.0]hexane (2.04 g,23.7 mol) (Barili, P. L.; Berti, G.; Mastrorilli, E.; Tetrahedron 1993,49, 6263) in EtOH (3 mL) at 90° C. for 2 d. Chromatography of the crudeproduct with 100% CH₂Cl₂ followed by 40:0.95:0.05-10:0.95:0.05CH₂Cl₂:MeOH:NH₄OH gave the product (615 mg, 87%) as a yellow solid: MSm/z 297 (M+H)⁺.

7.1.3 Step B: Preparation of(±)-trans-4-[4-(4-chlorobenzyl)-piperazin-1-yl]-tetrahydrofuran-3-ylamine

Following General Procedure B,(±)-trans-4-[4-(4-chlorobenzyl)-piperazin-1-yl]-tetrahydrofuran-3-ol(600 mg, 2.02 mmol) in CH₂Cl₂ (10 mL) was treated with Et₃N (0.56 mL,4.0 mmol) and MeSO₂Cl (0.31 mL, 4.0 mol) for 2 h and the resultantproduct was heated in dioxane (11.6 mL) and NH₄OH (4.7 mL) for 6 h.Chromatography of the crude product with 100% CH₂Cl₂ followed by30:0.95:0.05-10:0.95:0.05 CH₂Cl₂:MeOH:NH₄OH gave the product (330 mg,55%) as a yellow oil: MS m/z 296 (M+H)⁺.

Example 3

Preparation ofN-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-4-methyl-benzenesulfonamide

2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine (45 mg) wasdissolved in a solution of triethylamine in dichloromethane (3 mL, 0.29M in triethylamine). This solution was added to a test tube containingp-toluenesulfonyl chloride (60 mg). After standing at ambienttemperature for several days, the reaction was directly passed through asmall pad of silica and eluted with 10% ethyl acetate in hexanes, thenwith 50% ethyl acetate in hexanes. Solvent removal left 57.5 mg of theproduct: MS m/z 489 (M)⁺.

Example 4

Preparation of1-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-3-(3-methoxy-phenyl)-urea

2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine (45 mg) wasdissolved in a solution of triethylamine in dichloromethane (3 mL, 0.29M in triethylamine). This solution was added to a test tube containing3-methoxyphenyl isocyanate (45 mg). After standing at ambienttemperature for several days, the reaction was directly passed through asmall pad of silica and eluted with 10% ethyl acetate in hexanes, thenwith 50% ethyl acetate in hexanes. Solvent removal left 26.2 mg of theproduct, which was subsequently repurified by silica gel chromatographyto afford 9.4 mg product: MS m/z 484 (M)⁺.

Example 5

Preparation ofN-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-2-thiophen-2-yl-acetamide

2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine (45 mg) wasdissolved in a solution of triethylamine in dichloromethane (3 mL, 0.29M in triethylamine). This solution was added to a test tube containing2-thiopheneacetyl chloride (50 mg). After standing at ambienttemperature for several days, the reaction was directly passed through asmall pad of silica and eluted with 10% ethyl acetate in hexanes, thenwith 50% ethyl acetate in hexanes. Solvent removal left 48.6 mg of theproduct: MS m/z 459 (M)⁺.

Example 6

Preparation of4-chloro-N-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-4-methyl-benzamide

2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine (45 mg) wasdissolved in a solution of triethylamine in dichloromethane (3 mL, 0.29M in triethylamine). This solution was added to a test tube containing4-chlorobenzoyl chloride (60 mg). After standing at ambient temperaturefor several days, the reaction was directly passed through a small padof silica and eluted with 10% ethyl acetate in hexanes, then with 50%ethyl acetate in hexanes. Solvent removal left 49.6 mg of the product,which was subsequently repurified by silica gel chromatography to afford11.4 mg product: MS m/z 473 (M)⁺.

Example 7

Preparation ofN-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-4-methyl-benzamide

2-[4-(3,4-dichlorobenzyl)-piperazin-1-yl]-phenylamine (45 mg) wasdissolved in a solution of triethylamine in dichloromethane (3 mL, 0.29M in triethylamine). This solution was added to a test tube containing4-methylbenzoyl chloride (55 mg). After standing at ambient temperaturefor several days, the reaction was directly passed through a small padof silica and eluted with 10% ethyl acetate in hexanes, then with 50%ethyl acetate in hexanes. Solvent removal left 10.7 mg of the product:MS m/z 453 (M)⁺.

Example 8

Formulation Examples

The following are representative pharmaceutical Formulations containinga compound of Formula (I).

Tablet Formulation

-   -   The following ingredients are mixed intimately and pressed into        single scored tablets.

Quantity per Ingredient tablet, mg compound of this invention 400cornstarch 50 croscarmellose sodium 25 lactose 120 magnesium stearate 5Capsule Formulation

-   -   The following ingredients are mixed intimately and loaded into a        hard-shell gelatin capsule.

Quantity per Ingredient capsule, mg compound of this invention 200lactose, spray-dried 148 magnesium stearate 2Suspension Formulation

-   -   The following ingredients are mixed to form a suspension for        oral administration.

Ingredient Amount compound of this invention 1.0 g fumaric acid 0.5 gsodium chloride 2.0 g methyl paraben 0.15 g propyl paraben 0.05 ggranulated sugar 25.5 g sorbit (70% solution) 12.85 g Veegum K(Vanderbilt Co.) 1.0 g flavoring 0.035 ml colorings 0.5 mg distilledwater q.s. to 100 mlInjectable Formulation

-   -   The following ingredients are mixed to form an injectable        Formulation.

Ingredient Amount compound of this invention 0.2 g sodium acetate buffersolution, 0.4 M 2.0 ml HCl (1N) or NaOH (1N) q.s. to suitable pH water(distilled, sterile) q.s. to 20 mlLiposomal Formulation

-   -   The following ingredients are mixed to form a liposomal        Formulation.

Ingredient Amount compound of this invention  10 mgL-.alpha.-phosphatidylcholine 150 mg tert-butanol  4 ml

-   -   Freeze dry the sample and lyopholize overnight. Reconstitute the        sample with 1 ml 0.9% saline solution. Liposome size can be        reduced by sonication.

Example 9

CCR-3 Receptor Binding Assay—In Vitro

The CCR-3 antagonistic activity of the compounds of the invention wasdetermined by their ability to inhibit the binding of ¹²⁵I eotaxin toCCR-3 L1.2 transfectant cells (see Ponath, P. D. et al., J. Exp. Med.,Vol. 183, 2437-2448, (1996)).

The assay was performed in Costar 96-well polypropylene round bottomplates. Test compounds were dissolved in DMSO and then diluted withbinding buffer (50 mM HEPES, 1 mM CaCl.sub.2, 5 mM MgCl₂, 0.5% bovineserum albumin (BSA), 0.02% sodium azide, pH 7.24) such that the finalDMSO concentration was 2%. 25 μl of the test solution or only bufferwith DMSO (control samples) was added to each well, followed by theaddition of 25 μl of ¹²⁵I-eotaxin (100 pmol) (NEX314, New EnglandNuclear, Boston, Mass.) and 1.5×10⁵ of the CCR-3 L1.2 transfected cellsin 25 μl binding buffer. The final reaction volume was 75 μl.

After incubating the reaction mixture for 1 hour at room temperature,the reaction was terminated by filtering the reaction mixture throughpolyethylenimine treated Packard Unifilter GF/C filter plate (Packard,Chicago, Ill.). The filters were washed four times with ice cold washbuffer containing 10 mm HEPES and 0.5M sodium chloride (pH 7.2) anddried at 65° C. for approximately 10 minutes. 25 μl/well ofMicroscint-20® scintillation fluid (Packard) was added and theradioactivity retained on the filters was determined by using thePackard TopCount®

Compounds of this invention were active in this assay. Representativevalues for some of the compounds listed in Table 1 are shown below.

Cpd. No. IC50 (μM) 1 200 2 36 3 12.75 7 0.1099

Example 10

Inhibition of Eotaxin Mediated Chemotaxis of CCR-3 L1.2 TransfectantCells—In Vitro Assay

The CCR-3 antagonistic activity of the compounds of this invention canbe determined by measuring the inhibition of eotaxin mediated chemotaxisof the CCR-3 L1.2 transfectant cells, using a slight modification of themethod described in Ponath, P. D. et al., J. Clin. Invest. 97: 604-612(1996). The assay is performed in a 24-well chemotaxis plate (CostarCorp., Cambridge, Mass.). CCR-3 L1.2 transfectant cells are grown inculture medium containing RPMI 1640, 10% Hyclone® fetal calf serum, 55mM 2-mercaptoethanol and Geneticin 418 (0.8 mg/ml). 18-24 hours beforethe assay, the transfected cells are treated with n-butyric acid at afinal concentration of 5 mM/1×10⁶ cells/ml, isolated and resuspended at1×10⁷ cells/ml in assay medium containing equal parts of RPMI 1640 andMedium 199 (M 199) with 0.5% bovine serum albumin.

Human eotaxin suspended in phosphate buffered saline at 1 mg/ml is addedto bottom chamber in a final concentration of 100 nm. Transwell cultureinserts (Costar Corp., Cambridge, Mass.) having 3 micron pore size areinserted into each well and L1.2 cells (1×10⁶) are added to the topchamber in a final volume of 100 μl. Test compounds in DMSO are addedboth to the top and bottom chambers such that the final DMSO volume is0.5%. The assay is performed against two sets of controls. The positivecontrol contained cells with no test compound in the top chamber andonly eotaxin in the lower chamber. The negative control contains cellswith no test compound in the top chamber and neither eotaxin nor testcompound in lower chamber. The plate is incubated at 37° C. After 4hours, the inserts are removed from the chambers and the cells that havemigrated to the bottom chamber are counted by pipetting out 500 μl ofthe cell suspension from the lower chamber to 1.2 ml Cluster tubes(Costar) and counting them on a FACS for 30 seconds.

Example 11

Inhibition of Eotaxin Mediated Chemotaxis of Human Eosinophils—In VitroAssay

The ability of compounds of the invention to inhibit eotaxin mediatedchemotaxis of human eosinophils can be assessed using a slightmodification of procedure described in Carr, M. W. et al., Proc. Natl.Acad. Sci. USA, 91: 3652-3656 (1994). Experiments are performed using 24well chemotaxis plates (Costar Corp., Cambridge, Mass.). Eosinophils areisolated from blood using the procedure described in PCT Application,Publication No. WO 96/22371. The endothelial cells used are theendothelial cell line ECV 304 obtained from European Collection ofAnimal Cell Cultures (Porton Down, Salisbury, U.K.). Endothelial cellsare cultured on 6.5 mm diameter Biocoat.™. Transwell tissue cultureinserts (Costar Corp., Cambridge, Mass.) with a 3.0 μM pore size.Culture media for ECV 304 cells consists of M199, 10% Fetal Calf Serum,L-glutamine and antibiotics. Assay media consists of equal parts RPMI1640 and M199, with 0.5% BSA. 24 hours before the assay 2×10⁵ ECV 304cells are plated on each insert of the 24-well chemotaxis plate andincubated at 37° C. 20 nM of eotaxin diluted in assay medium is added tothe bottom chamber. The final volume in bottom chamber is 600 μl. Theendothelial coated tissue culture inserts are inserted into each well.10⁶ eosinophil cells suspended in 100 μl assay buffer are added to thetop chamber. Test compounds dissolved in DMSO are added to both top andbottom chambers such that the final DMSO volume in each well was 0.5%. The assay is performed against two sets of controls. The positive controlcontains cells in the top chamber and eotaxin in the lower chamber. Thenegative control contains cells in the top chamber and only assay bufferin the lower chamber. The plates are incubated at 37° C. in 5% CO₂/95%air for 1-1.5 hours.

The cells that migrate to the bottom chamber are counted using flowcytometry. 500 μl of the cell suspension from the lower chamber areplaced in a tube, and relative cell counts are obtained by acquiringevents for a set time period of 30 seconds.

Example 12

Inhibition of Eosinophil Influx into the Lungs of Ovalbumin SensitizedBalb/c Mice by CCR-3 Antagonist—In Vivo Assay

The ability of the compounds of the invention to inhibit leukocyteinfiltration into the lungs can be determined by measuring theinhibition of eosinophil accumulation into the bronchioalveolar lavage(BAL) fluid of Ovalbumin (OA)-sensitized balb/c mice after antigenchallenge by aerosol. Briefly, male balb/c mice weighing 20-25g aresensitized with OA (10 μg in 0.2 ml aluminum hydroxide solution)intraperitoneally on days 1 and 14. After a week, the mice are dividedinto ten groups. Test compound or only vehicle (control group) oranti-eotaxin antibody (positive control group) is administered eitherintraperitoneally, subcutaneously or orally. After 1 hour, the mice areplaced in a Plexiglass box and exposed to OA aerosol generated by aPARISTAR.™. nebulizer (PARI, Richmond, Va.) for 20 minutes. Mice whichhave not been sensitized or challenged are included as a negativecontrol. After 24 or 72 hours, the mice are anesthetized (urethane,approx. 1 g/kg, i.p.), a tracheal cannula (PE 60 tubing) is inserted andthe lungs are lavaged four times with 0.3 ml PBS. The BAL fluid istransferred into plastic tubes and kept on ice. Total leukocytes in a 20μl aliquot of the BAL fluid is determined by Coulter Counter.™.(Coulter, Miami, Fla.). Differential leukocyte counts are made onCytospin.™. preparations which have been stained with a modifiedWright's stain (DiffQuick.™.) by light microscopy using standardmorphological criteria.

The foregoing invention has been described in some detail by way ofillustration and example, for purposes of clarity and understanding. Itwill be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

1. A compound of Formula (I):

wherein: R¹ is (C₁-C₂)alkylene; R² is a phenyl group which is optionallysubstituted with one or more substituents selected from the groupconsisting of alkyl, haloalkyl, hydroxyalkyl, heteroalkyl, acyl,acylamino, amino, alkylamino, dialkylamino, alkylthio, alkylsulfinyl,alkylsulfonyl, —SO₂NR′R″ (where R′ and R″ are independently hydrogen oralkyl), alkoxy, haloalkoxy, alkoxycarbonyl, carbamoyl, hydroxy, halo,nitro, cyano, mercapto, methylenedioxy or ethylenedioxy; R³ is hydrogen,alkyl, acyl, aryl, or arylalkyl; ring A is a heterocyclyl, or optionallya phenyl group which is optionally substituted with one or moresubstituents selected from the group consisting of alkyl, haloalkyl,hydroxyalkyl, heteroalkyl, acyl, acylamino, amino, alkylamino,dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, —SO₂NR′R″ (whereR′ and R″ are independently hydrogen or alkyl), alkoxy, haloalkoxy,alkoxycarbonyl, carbamoyl, hydroxy, halo, nitro, cyano, mercapto,methylenedioxy or ethylenedioxy; L is —C(═O)—, —C(═S)—, —SO₂—,—C(═O)N(R_(a))—, —C(═S)N(R_(a))—, —SO₂N(R_(a))—, —C(═O)O—, —C(═S)O—,—S(═O)₂O—; where R_(a) is hydrogen, alkyl, acyl, aryl, arylalkyl,alkoxycarbonyl, or benzyloxycarbonyl; X is absent, —(CR′R″)O—,—(CR′R″)S—, —(CR′R″)NR_(b)— or alkylene; where R′ and R″ areindependently hydrogen or alkyl, and R_(b) is hydrogen or alkyl; R⁴ isaryl or heteroaryl; and prodrugs, individual isomers, racemic andnon-racemic mixtures of isomers, and pharmaceutically acceptable saltsand solvates thereof.
 2. The compound of claim 1, which is a compound ofFormula (II):

wherein R¹-R⁴, A, L, and X have any of the values described in claim 1.3. The compound of claim 1, which is a compound of Formula (III):

wherein R¹-R⁴, A, L, and X have any of the values described in claim 1.4. The compound of any one of claims 1-3 wherein R¹ is methylene.
 5. Thecompound of any one of claims 1-3 wherein R² is 4-chlorophenyl or3,4-dichlorophenyl.
 6. The compound of any one of claims 1-3 wherein R³is hydrogen.
 7. The compound of any one of claims 1-3 wherein L is—C(═O)—, —SO₂—, —C(═O)N(R_(a))—, —C(═S)N(R_(a))—, or —C(═O)O—.
 8. Thecompound of any one of claims 1-3 wherein L is —C(═O)—.
 9. The compoundof any one of claims 1-3 wherein L is —C(═O)N(R_(a))—.
 10. The compoundof claim 1 which is a compound of formula (IV):

wherein R³, R⁴, A, L, and X have any of the values described in claim 1.11. The compound of claim 1 which is a compound of formula (VIII):

wherein X and R⁴ have any of the values defined in claim 1; and R^(x) ishydrogen, alkyl, acyl, alkylsulfonyl, aminosulfonyl,(alkylamino)sulfonyl, (dialkylamino)sulfonyl, carbamoyl,(alkylamino)carbonyl, (dialkylamino)carbonyl, (carbamoyl)alkyl,(alkylamino)carbonylalkyl, or dialkylaminocarbonylalkyl.
 12. Thecompound of claim 1 which is a compound of formula (IX):

wherein X and R⁴ have any of the values defined in claim
 1. 13. Thecompound of claim 1 which is a compound of formula (X):

wherein X and R⁴ have any of the values defined in claim
 1. 14. Thecompound of claim 7 wherein X is absent, methylene, 1,2-ethanediyl,1,3-propanediyl, or 1,4-butanediyl.
 15. The compound of claim 14 whereinR⁴ is 3,4-dichlorophenyl, 3,4,5-trimethoxyphenyl,4-methanesulfonylphenyl, 3-methanesulfonylphenyl,4-methoxynaphthalen-2-yl, 5-(3,4-dimethoxyphenyl)pyrimidin-2-yl, phenyl,3-fluorophenyl, 4-ethylphenyl, 3-methoxyphenyl, 2,4-difluorophenyl,3-trifluoromethylphenyl, 4-methylphenyl, 4-fluorophenyl, 2-fluorophenyl,4-carboxamidophenyl, 4-acetylphenyl, 4-nitrophenyl, 2-methylphenyl,2-chloro-4-fluorophenyl, 3,4-dimethoxyphenyl, 2,5-dimethoxyphenyl,2,3-dichlorophenyl, 2,4-dichlorophenyl, 4-bromophenyl,4-chloro-3-nitrophenyl, 2-nitrophenyl, 2-nitro-4-trifluoromethylphenyl,4-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 3-methylphenyl,2-trifluoromethylphenyl, 2-methoxyphenyl, 3-bromophenyl,4-trifluoromethylphenyl, 3-trifluoromethylphenyl,3,5-bis-trifluoromethylphenyl, 4-tert-butylphenyl, 4-ethoxyphenyl,3-cyanophenyl, 4-cyanophenyl, 4-methoxyphenyl, 3-nitrophenyl,3,5-dimethoxyphenyl, 4-iodophenyl, 4-isopropylphenyl,3-methoxycarbonylphenyl, 3-acetylphenyl, 2-methylphenyl, indol-2-yl,5-methoxyindol-2-yl, 5-chloroindol-2-yl, 2-methoxycarbonylphenyl,3,5-dichlorophenyl, 1-naphthyl, 3-chloro-2-methylphenyl,2,5-dimethylphenyl, 2-thienyl, 3-ethoxyphenyl, 3-isoquinolyl,2-methylquinolin-6-yl, 3-methylaminophenyl, 3-quinolyl, 2-quinolyl,5-hydroxynaphthalen-2-yl, 8-hydroxyquinolin-2-yl,5,7-dimethyl-[1,8]naphthyridin-2-yl, 6-quinolyl, 3-(acetylamino)phenyl,or 2,3,4-trimethoxyphenyl.
 16. The compound of claim 15 wherein R⁴ is3,4,5-trimethoxyphenyl, 4-acetyl-phenyl, 3-carboxamidophenyl,4-carboxamidophenyl, 3-methanesulfonylphenyl or 4-methanesulfonylphenyl.17. The compoundN-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-2-thiophen-2-yl-acetamide;N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-3,4-difluoro-benzamide;1-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-3-(3-methoxy-phenyl)-urea;4-Chloro-N-{2-[4-(3,4-dichloro-benzyl)-piperazin-1-yl]-phenyl}-benzamide;N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-4-methyl-benzenesulfonamide;1-{4-[4-(4-Chloro-benzyl)-piperazin-1-yl]-tetrahydrofuran-3-yl}-3-(3,4,5-trimethoxy-phenyl)-urea;N-{2-[4-(3,4-Dichloro-benzyl)-piperazin-1-yl]-phenyl}-4-methyl-benzamide;or a salt thereof.
 18. A composition containing a therapeuticallyeffective amount of a compound as described in any one of claims 1-10,or a salt thereof; and an excipient.
 19. A method of treatment of adisease in a mammal treatable by administration of a CCR-3 receptorantagonist, comprising administering to the mammal a therapeuticallyeffective amount of a compound of Formula I as described in any one ofclaims 11-17, or a salt thereof.
 20. The method of claim 19 wherein thedisease is asthma.